NIH Public Access Author Manuscript Best Pract Res Clin Obstet Gynaecol

NIH Public Access Author Manuscript Best Pract Res Clin Obstet Gynaecol. Author manuscript; available in PMC 2013 May 23.

NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Best Pract Res Clin Obstet Gynaecol. 2012 August ; 26(4): 451–462. doi:10.1016/j.bpobgyn.2012.01.004.

Formulation, pharmacokinetics and pharmacodynamics of topical microbicides

Jessica L. Adams, PharmD, BCPSa,* [HIV Pharmacology Fellow] and Angela D.M. Kashuba, BScPhm, PharmD, DABCPb [Professor and Director] aUniversity of North Carolina at Chapel Hill, Eshelman School of Pharmacy 3315 Kerr Hall CB# 7569, Chapel Hill, North Caroina 27599-7569, USA bDivision of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, UNC Center for AIDS Research Clinical Pharmacology and Analytical Chemistry Core, University of North Carolina at Chapel Hill, North Carolina, USA

Abstract The development of safe topical microbicides that effectively prevent human immunodeficiency virus (HIV) infection is a major goal in curbing the human immunodeficiency virus pandemic. A number of past failures resulting from mucosal toxicity or lack of efficacy have informed the field. Products that caused toxicity to the female genital tract mucosa, and thereby increased the likelihood of HIV acquisition, included nonoxynol 9, cellulose sulfate, and C31 G vaginal gel Savvy®. Topical products that were ineffective in preventing HIV infection include BufferGel®, Carraguard®, and PRO 2000®. Antiretroviral drugs such as tenofovir and dapivirine formulated into microbicide products have shown promise, but there is much to learn about ideal product formulation and acceptability, and drug distribution and disposition (pharmacokinetics). Current formulations for water-soluble molecules include vaginally or rectally applied gels, vaginal rings, films and tablets. Dosing strategies (e.g. coitally dependent or independent) will be based on the pharmacokinetics of the active ingredient and the tolerance for less than perfect adherence.

Keywords microbicide; pharmacokinetics; pharmacodynamics; gel; ring; film; pre-exposure prophylaxis

Introduction In order to be effective, an ideal microbicide should not cause mucosal irritation, should distribute into the tissues that are associated with HIV transmission, be metabolised efficiently to their active components (if necessary), and achieve adequate and sustained concentrations for protection against human immunodeficiency virus (HIV) transmission. Absorption into the systemic circulation, and subsequent exposure in blood plasma, should be minimal to diminish systemic adverse effects and the potential for the virus to become drug resistant if HIV infection should occur. A microbicide must also be easy to use and well accepted by the target population. In this chapter, we discuss the lessons learned from past microbicide failures, review successes and outline current knowledge of microbicide formulations in development.

© 2012 Elsevier Ltd. All rights reserved. *Corresponding author. Tel.: +1 919 962 0029; Fax: +1 919 962 0644. [email protected] (J.L. Adams). Conflict of interest None declared. Adams and Kashuba Page 2

Microbicide formulation Product considerations NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Microbicide vehicles are important, as they allow the active drug to reach the proper site of activity, to distribute throughout the luminal surface (vagina or colorectum), and remain at the surfaces for an adequate (as yet undefined) period of time. Vehicles can include aqueous gels, reservoir rings, and locally applied solid films and tablets.

Physiochemical characteristics of a gel vehicle that must be optimised include volume, viscosity, yield stress, sheer rate, pH and osmolality.1 Reservoir rings must be formulated to provide long-term, predictable, sustained release of an active component throughout the vaginal compartment.2 Film polymers should be non-toxic, non-irritant, have adequate wetting, spreadability, disintegration over time, and strength. Solid vaginal tablets must also have adequate disintegration and dispersion throughout the vaginal compartment without causing irritation.1 For all formulations, it is important that the pharmaceutically active compound be stable in the vehicle in which it is formulated, and uniformly distributed to allow for broad distribution in the vaginal compartment. A full list of the advantages and disadvantage of each of the formulations in development is presented in Table 1.

Toxicity to vaginal mucosa Above all, the field has learned that microbicides must demonstrate no mucosal toxicity, which can lead to irritation, epithelial disruption, release of cytokines, immune activation and changes to flora, which increase the risk of HIV acquisition. In order for a product to be compatible with the mucosal environment, physiologic pH must be maintained and the product should be iso-osmolar.3 Past microbicide gel failures owing to local mucosal toxicity included those containing nonoxynol 9,4 cellulose sulfate,5 and Savvy®.6 The following is a summary of the findings from those studies.

Nonoxynol-9 COL-1492 was a phase III, randomised, placebo-controlled study in 765 female sex workers using 1.5 g of carbomer gel (a polymer with bio-adhesive properties), containing 3.5% nonoxynol-9 (a spermicidal surfactant).4 A 16% seroconversion rate was seen in the active arm of the study, compared with 12% in the placebo arm. Women who used the product more frequently had higher rates of HIV infection and were found more likely to have lesions with epithelial disruption. Those women reporting a mean applicator use greater than 3.5 per working day had a hazard ratio of 1.8 (1.0 to 3.2) for HIV incidence (P = 0.03) compared with placebo. Subsequently, it was determined that nonoxynol-9 causes cytokine release, immune-cell recruitment, and alterations in vaginal flora, all of which may make women more susceptible to HIV acquisition.7 In particular, release of the cytokine interleukin-1 activates nuclear factor-kappa B, leading to chemokine-induced immune cell recruitment, which provided host cells for HIV-1 infection and increased HIV replication.8 In another study,9 after the use of nonoxynol-9 containing diaphragms, women had vaginal colonisation of Escherichia coli, Enterococcus species, and anaerobic gram-negative rods, which could also lead to immune activation and immune-cell recruitment.

Cellulose sulfate Cellulose sulfate is a polyanion entry inhibitor with in-vitro activity against HIV-1 and HIV-2, as well as Neisseria gonorrhoeae, and Chlamydia trachomatis.10 It was found to be well tolerated in early phase safety studies using single and multiple daily doses for up to 2 weeks.11–14 A phase III study compared 3.5 ml of 6% cellulose sulfate gel with placebo gel in 1398 women 1 h before intercourse. Interim analysis found 24 seroconversions in the

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cellulose sulfate arm and 11 in the placebo arm (hazard ratio 2.23; P = 0.02) leading to premature discontinuation of the study. Final analysis revealed 25 out of 706

NIH-PA Author Manuscript NIH-PA Author Manuscriptseroconversions NIH-PA Author Manuscript in the cellulose sulfate arm and 16 out of 692 in the placebo arm (hazard ratio 1.61; P = 0.13).5 Possible explanations of the results include increased inflammatory reactions and immune cell recruitment.15 Although not statistically significant, one of the safety studies had shown slightly more women with signs of increased inflammation on colposcopy after using cellulose sulfate gel twice daily for 14 days.16

SAVVY® The chemical compound C31 G (SAVVY®) is a surfactant that disrupts the outer membrane of HIV with a similar mechanism to nonoxynol-9.17 In multiple phase I studies, signs of irritation with repeat dosing of 1% SAVVY® were reported, but considered less toxic than nonoxynol 9.18–20 One colposcopy study found 1.2% SAVVY® to cause similar epithelial disruption as 2% nonoxynol-9.21 Subsequently, a phase III study6 enrolling 2153 women using 3.5 ml of product in a coitally dependent manner observed more seroconversions in the active arm (21 out of 1076) than in the placebo arm (12 out of 1077). The study was stopped early because of an overall lower than expected HIV incidence: mid-point analysis determined that an additional 1980 participants would be needed to reach the overall 66 events required to show efficacy. The true hazard ratio and reasons for the increased number of infections in the SAVVY® arm could not be determined. An additional parallel phase III study of 2142 women in Ghana was also stopped early because of a lower than expected HIV incidence rate.22

Vivagel® Vaginal irritation and inflammation was found to occur when 3% Vivagel® (SPL 7013) was used twice daily for 14 days. Two out of 35 women discontinued the gel as a result of genital irritation, confirmed on colposcopy.23 A second study of 3% Vivagel®24 evaluated inflammatory markers in the vaginal mucosa of women using the gel twice daily for 14 days. By day 7, interleukin-6, CD8+ and CD69+ cells, CD4+ and CD69+ cells and CD4+ and CCR5+ cells were all elevated (P < 0.05) in women using the gel compared with placebo. By day 14, interferon gamma, interleukin-5 and interleukin-10 (P = 0.001) were elevated. All were reversible with discontinuation of VivaGel®, and this product is continuing in Phase II studies for the treatment and prevention of bacterial vaginosis.25

Lack of efficacy A number of topical microbicides have failed to show efficacy at preventing HIV infection, including BufferGel®,26 PRO 2000®,27 and Carraguard®.28 By maintaining vaginal pH 4.0– 4.5 in the presence of semen, BufferGel® was intended to protect against HIV infection by broadly acidifying pathogens.29 A phase II/IIb, four-arm study in 3101 women comparing BufferGel® with 0.5% PRO 2000® gel, placebo gel, and no gel found no difference in HIV infection rates between BufferGel® and placebo gel (hazard ratio 1.10; P = 0.63), and BufferGel® and no gel (hazard ratio 1.05, P = 0.78).26 This same study showed modest benefit of a 0.5% PRO 2000® gel over placebo gel (hazard ratio 0.7, P = 0.10) and no gel (hazard ratio 0.67, P = 0.06).26

PRO 2000® gel is a synthetic naphthalene sulfonate polymer that inhibits viral attachment and entry. It demonstrated antiviral activity in macaques30 and in vitro.31 A subsequent phase III study27 showed that 2% and 0.5% of PRO 2000® gel were ineffective at preventing HIV infection compared with placebo. The 2% PRO 2000® gel arm was stopped prematurely due to futility, and the 0.5% PRO 2000® gel arm failed to show a difference in HIV incidence compared with placebo (hazard ratio 1.05; P = 0.71).

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Carraguard® contains the polyanion carrageenan. The mechanism is not fully understood, but it may bind the positively charged regions of the viral envelope and act as a barrier to 32

NIH-PA Author Manuscript NIH-PA Author Manuscripthost NIH-PA Author Manuscript cells. It was previously shown to prevent effectively HIV infection in vitro and in macaques.33 In a phase III study conducted in 6005 women in South Africa, 4 ml of 3% Carraguard® failed to demonstrate efficacy compared with placebo (covariate adjusted hazard ratio 0.87, 95% CI 0.69 to 1.09).28

Owing to the significant number of failures of surfactants, polyanions, and general antimicrobials, a number of small molecule antiretroviral drugs are under investigation. A review of the microbicide formulations that are currently under development follows. A summary of these data are presented in Table 2.

Microbicide gels Antiretroviral drugs formulated as gels show promise in preventing HIV infection. Gel formulations allow for the application of antiretroviral drugs directly to the vaginal mucosa with limited systemic absorption and side-effects, and HIV resistance potential. Currently, tenofovir with and without emtricitabine, and dapivirine are being investigated in gel formulations.

Tenofovir Tenofovir is a nucleotide reverse transcriptase inhibitor used orally in combination for the treatment of HIV-1.34 It acts as a nucleoside analogue that terminates HIV transcription from RNA to DNA before integration into the host cell genome.34 By targeting a place in the HIV life cycle before genome integration, tenofovir could theoretically prevent initial cellular infection. Tenofovir gel has been tested in macaques, humanised mice, and in clinical studies.

Tenofovir 1% gel has been tested in macaques, with repeated exposure to the chimeric simian-human immunodeficiency virus (SHIV). In one study, 3 ml tenofovir 1% gel given vaginally to female macaques twice weekly before SHIV challenge for a total of 20 challenges protected six out of six macaques from infection.35 Additionally, tenofovir 1% gel protected four out of six macaques against SHIV challenge 3 days after gel application.36 Rectally applied 1% tenofovir gel was shown to protect eight out of nine macaques when applied 2 h before simian immunodeficiency virus exposure.37 Pharmacokinetic data of both vaginal and rectally applied 1% tenofovir gel in macaques showed quick and thorough distribution throughout mucosal surfaces, with concentrations 4–5 logs higher in the mucosal compartment than in blood plasma.38

In humanised mice, topically applied tenofovir 1% gel was found to be 88% effective (P = 0.002) at preventing HIV infection compared with placebo gel when 20 μl of gel was applied vaginally 4 h before and after HIV challenge.39

A phase I pharmacokinetic study in humans assessed 2 weeks of single and multiple dosing of once or twice daily 4 ml applications of 1% tenofovir vaginal gel in blood plasma, endocervical cells, cervicovaginal fluid aspirates, and vaginal tissue in 49 women from the Dominican Republic and two US sites.40 Tenofovir achieved high persistent concentrations within the vaginal lumen and tissues, and minimal concentrations in the systemic circulation. No differences between proximal and distal tissue concentrations were found, demonstrating uniform distribution throughout the vagina. Cervicovaginal fluid concentrations were greater than 1,000,000 ng/ml within the first 4 h of dosing, and fell to 100,000 ng/ml by 24 h. Tenofovir vaginal tissue concentrations were found to be approximately one to two logs (10 to 100 times) lower than luminal concentrations. About

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3–5% of the tenofovir was metabolised intracellularly to the active metabolite tenofovir diphosphate, in vaginal and cervical cells within 30 mins of dosing. Blood plasma

NIH-PA Author Manuscript NIH-PA Author Manuscriptconcentrations NIH-PA Author Manuscript after vaginal application were less than 5% of those previously reported after oral dosing.41 These data were subsequently confirmed in an additional study using an African population.42

The vaginally formulated 1% tenofovir gel was assessed in a phase I placebo-controlled crossover study investigating rectal administration compared with oral administration of tenofovir disoproxyl fumarate.43 Eighteen healthy adults sequentially received a single dose of oral tenofovir disoproxyl fumarate 300 mg, then either 1% tenofovir gel applied rectally or a placebo gel. Rectal dosing resulted in 30-fold lower plasma exposure than oral dosing. Ex-vivo HIV challenges were carried out on rectal biopsies obtained after 7 days of daily rectal gel use. Concentrations of the active form of the drug, intracellular tenofovir diphosphate, greater than 1000 fmol/mg were required to see significant suppression of viral replication in these tissues. As significant gastrointestinal discomfort was seen with the vaginal formulation of this gel, a rectal-specific gel with decreased osmolality has been formulated and is undergoing clinical safety and efficacy studies.44,45

Tenofovir 1% gel was the first antiretroviral to demonstrate its ability to prevent the sexual transmission of HIV.46 In the CAPRISA 004 study conducted in rural and urban sites in South Africa,47 843 women were instructed to vaginally apply 4 ml of 1% tenofovir gel up to 12 h before and after sex with no more than two doses used in 24 h. This method was proven to be 39% effective at preventing HIV infection compared with placebo overall. In women with over 80% adherence (as assessed by used applicator counts), efficacy was 54%. Pharmacological analysis of cervicovaginal fluid samples found a direct correlation between higher tenofovir concentrations and protection from both HIV infection and herpes simplex 2 infection (a surprising and unexpected benefit). Kaplan–Meier analysis showed that women with cervicovaginal fluid concentrations of tenofovir less than 1000 ng/ml had an HIV incidence rate similar to that of women using placebo. Women with cervicovaginal fluid concentrations greater than 1000 ng/ml had a significantly lower chance of acquiring HIV.48

The VOICE (Vaginal plus Oral Interventions to Control the Epidemic; MTN 003) study49 was designed to compare the efficacy of daily dosed 1% vaginal gel, a placebo gel, oral tenofovir, oral tenofovir plus emtricitabine and an oral placebo. In November 2011, the daily 1% tenofovir gel arm of the study was halted due to futility. With about 1000 women enrolled in each arm, a review of the data from September 9, 2011 to September 30, 2011 found a 6.1% infection rate in the placebo gel arm and a 6.0% infection rate in the tenofovir gel arm. The reasons for the failure of this VOICE arm after the 39% success rate of CAPRISA 004 have yet to be determined. Limited adherence to the daily dosing of tenofovir 1% gel or increased drug exposures with the CAPRISA 004 strategy may have been factors. One additional phase III study of 1% tenofovir vaginal gel called FACTS 001 (Follow-on African Consortium for Tenofovir Studies) is currently ongoing. FACTS 001 is evaluating the effectiveness of the CAPRISA 004 dosing strategy in 2200 women in South Africa.50

Dapivirine Because of its poor oral bioavailability, dapivirine (TMC120) is being developed solely as a topical non-nucleoside reverse transcriptase inhibitor for the prevention of HIV infection. Dapivirine allosterically blocks the reverse transcriptase enzyme and thereby prevents the complete transcription of HIV RNA to DNA.51 Dapivirine’s tight binding to reverse transcriptase and high lipophilicity makes it active against both cell associated and cell free

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virus in semen without the metabolic activation that is needed for nucleotide reverse transcriptase inhibitors.52 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript In animal studies, a 0.009% dapivirine vaginal gel was given over 7 days to both rabbits (0.5 ml per day) and macaques (1 ml per day). Pharmacokinetic data in both species showed low plasma drug concentrations (maximum 0.225 ng/ml). Cervical and vaginal tissue concentrations were above the in-vitro 90% Effective Concentration (EC90) of 0.9 ng/ml for up to 48 h after dosing. Microautoradiography identified drug-related material on the surfaces of vaginal and cervical tissues, suggesting that the drug may reside in compromised areas of tissues and provide a protective effect at vulnerable lesions and ulcers.53,54

Dapivirine has shown to be safe and well tolerated with low systemic absorption in phase I and II studies investigating 2.5 ml doses of 0.001, 0.002, 0.005, and 0.02% dapivirine vaginal gel given twice daily for 42 days to 119 women. Plasma concentrations were dose proportional over the range studied, with no accumulation past 7 days of dosing. The maximum observed plasma concentration was a low 474 pg/ml in the 0.02% dose group.55

Dapivirine concentrations in cervicovaginal fluids and tissues were found to be well distributed throughout the vaginal compartment over 10 days of gel dosing in 18 healthy women. Women applied 2.5 ml of either 0.001%, 0.005% or 0.02% dapivirine gel once daily on days 1 and 10 and twice daily on days 2 to 9. Fluid concentrations were found to be 4.6–8.3 × 106 pg/ml on day 1 and 2.3–20.7 × 106 pg/ml on day 10, and cervicovaginal tissue biopsies collected on day 10 had concentrations of 1.0–356 × 103 pg/mg.56

Recently, a phase I study57 evaluated two different formulations of 0.05% dapivirine gel for pharmacokinetics and safety. The gels were applied daily for 11 days, and both were safe and well tolerated. The maximal dapivirine plasma concentrations did not exceed 1.1 ng/ml. The cervicovaginal fluid concentrations were 5 logs higher than dapivirine’s in-vitro IC50 for wild type HIV-1 virus and 3 to 5 logs higher than the in-vitro IC50 for virus containing the non-nucleoside reverse transcriptase inhibitor resistance mutation Y181C. The terminal half-life was 16–17 h. The dapivirine gel is currently undergoing additional phase I and II studies.58

Combination gel products Combinations of drugs formulated into a gel have a number of theoretical advantages. Combining drugs with different resistance profiles may protect against transmission of a virus with mutations to one of the agents, and may protect against rapid development of resistance if infection occurs.1 Additionally, combining drugs that act synergistically or additively may allow for lower doses of agents used in combination than used individually, thereby minimising systemic exposure and adverse effects.

Combinations currently under investigation include tenofovir with emtricitabine, and tenofovir with dapivirine.58 Macaques given 3 ml of gel combining 1% tenofovir with 5% emtricitabine were all protected against SHIV infection. Only 0.03% of the tenofovir dose and 0.025% of the emtricitabine dose were systemically absorbed 30 mins after gel application.35

In-vitro data suggest that dapivirine combined with tenofovir may be synergistic against transmitted viruses containing a particular non-nucleoside reverse transcriptase inhibitor mutation (Y181C). This virus is easily transmitted because it confers little or no fitness cost.59 This study supports the hypothesis that combination microbicide products may retain antiviral activity against resistant viruses, thereby better protecting against the transmission of resistant viruses.60

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Microbicide rings Vaginal rings containing antiretroviral drugs are similar in concept to vaginal rings currently NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript used for contraception and hormone replacement therapy.2 The intention of the ring is to maintain long-term, sustained antiretroviral release for local effect. Owing to the long-term drug release, rings can be used in a coitally independent manner, and only inserted monthly. This may have an adherence benefit over other microbicides in development.

Dapivirine Dapivirine has been formulated into a ring currently undergoing phase III studies.58 Previously, two phase I studies evaluated dapivirine 25 mg and 200 mg delivered from a vaginal ring over 7 days in 25 healthy women. Both studies found the ring to be safe, and adverse effects similar to placebo. Mucosal fluid was sampled at 4 h, 24 h and 7 days after insertion in all women from the introitus, cervix and ring area, and mean drug concentrations exceeded 1000 times the EC50 against wild-type HIV-1. In both studies, the mean concentrations of dapivirine were 0.7 to 7.1 μg/ml in mucosal fluid, and ranged from 0.3 to 3.5 μg/g in cervical and vaginal tissues. The authors point out that the lowest concentration in any of the tissues sampled was over 30-fold higher than the in-vitro EC90. Plasma concentrations did not exceed 40 pg/ml with this device.61

A recent double-blind, randomised (3:1) placebo-controlled study in 48 women62 evaluated the pharmacokinetics of multiple dosing of a 25 mg dapivirine vaginal ring. Dapivirine concentrations and safety assessments were measured over a total of 88 days. Dapivirine concentrations peaked in vaginal fluid within the first 24 h after the ring was inserted. Concentrations above 0.3 ng/ml (IC50) were maintained up to 35 days. Minimum concentrations were 1.9, 1.4 and 0.7 μg/g measured near the ring, at the cervix and the introitus, respectively; maximum concentrations were 66.1, 58.4 and 40.1 μg/g. Maximal plasma concentrations were 553 pg/ml. This study verified both the safety of the dapivirine ring and the ability of this product to maintain consistent drug delivery over 35 days.

Combination rings A combination dapivirine and maraviroc ring is currently in phase 1.63 These two antiretroviral drugs work differently: dapivirine by allosterically inhibiting reverse transcriptase, and maraviroc by preventing HIV from binding to the cell surface co-receptor CCR5.64 A dapivirine plus maraviroc combination gel has also been formulated, but studies of the gel are on hold to focus on the development of the ring formulation.65 Additionally, combining antiretroviral drugs with contraceptives into a single vaginal ring may be beneficial for women who seek protection against HIV infection while also avoiding conception. One of the largest challenges to the development of a dual releasing vaginal ring is creating differing dissolution conditions for drug entities with disparate physicochemical properties.66

Microbicide films and tablets Intravaginal films and tablets are already used as antifungals, antibacterials, and hormone replacement products. Advantages of these dosage forms include their portability, ease of storage and ease of use. Additionally, solid dosage forms can stabilise drugs that would be otherwise degraded in an aqueous solution. Limitations include an increased risk of vaginal irritation and the need for vaginal hydration to assure uniform distribution and tissue absorption.1

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The retrocyclin 1 peptide analog, RC-101, is currently being studied in a vaginal film formulation. This entry inhibitor prevents the binding of HIV to both CXCR4 and CCR5 co- 67

NIH-PA Author Manuscript NIH-PA Author Manuscriptreceptors NIH-PA Author Manuscript located on CD4+ cells in vitro. RC-101 is best formulated in a non-aqueous solid film dosage form because the peptide is degraded in semisolid aqueous solutions.68,69 RC-101 was formulated into a quick-dissolving 2000 μg per film product and applied intravaginally to six female macaques daily for 4 days. Over this time, the peptide was detected in cytobrush and tissue samples, but not in cervicovaginal lavage or plasma. Cervical and vaginal tissues taken from two macaques one day after the last film application were protected from SHIV challenge in an ex-vivo culture system. Colposcopy revealed no mucosal changes. This study contributes to the continued development of RC-101 vaginal film.68

Other films and tablets are in early preclinical development, and include a film containing a combination of tenofovir with maraviroc,58 and a vaginal tablet containing BMS793, a gp120 mediated cell attachment inhibitor.58

Conclusion A major advantage to the development and use of locally applied microbicides to prevent HIV infection is the ability to preclude partner negotiation, as must occur with condoms. Various topical formulations are being developed to provide a variety of options for a safe, effective, and acceptable product that can protect against HIV infection. Regardless of potency, microbicides will only be effective if they are used properly and consistently.70 A successful product rests on the ability to safely achieve adequate concentrations to prevent HIV transmission in mucosal tissues, and to maintain those concentrations throughout the window of most risk for HIV acquisition. As shown by CAPRISA 004 and VOICE, the most appropriate dosing regimen to ensure adherence, while also maintaining adequate microbicide concentrations, is yet to be determined. Just as they have for treatment, combination regimens that target different steps in the HIV life-cycle may prove most effective for prevention.

Acknowledgments

Angela Kashuba and Jessica Adams are funded by NIH 1U01AI095031-01 and CFAR 5P30AI050410-13.

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*48. Abdool Karim SS, Kashuba ADM, Werner L, Abdool Karim Q. Drug concentrations after topical and oral antiretroviral pre-exposure prophylaxis: implications for HIV prevention in women. Lancet. 2011; 378:279–281. [PubMed: 21763939] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 49. Microbicide Trials Network. [accessed 15.01.12] MTN statement on decision to discontinue use of tenofovir gel in VOICE, a major HIV prevention study in women. Immediate press release 25 Nov 2011; http://www.mtnstopshiv.org/node/3909 50. FACTS Consortium. [accessed 15.01.12] FACTS 001 study design. http://www.facts- consortium.co.za/ 51. Das K, Clark AD Jr, Lewi PJ, et al. Roles of conformational and positional adaptability in structure-based design of TMC125-R165335 (etravirine) and related nonnucleoside reverse transcriptase inhibitors that are highly potent and effective against wild-type and drug-resistant HIV-1 variants. J Med Chem. 2004; 47:2550–2560. [PubMed: 15115397] 52. D’Cruz OJ, Uckun FM. Dawn of non-nucleoside inhibitor based anti-HIV microbicides. J Antimicrob Chemother. 2006; 57:411–423. [PubMed: 16431862] *53. Nicol MR, Kashuba ADM. Pharmacologic opportunities for HIV prevention. Clin Pharmacol Ther. 2010; 88:598–609. [PubMed: 20881955] 54. Nuttall JP, Thake DC, Lewis MG, et al. Concentrations of dapivirine in the rhesus macaque and rabbit following once daily intravaginal administration of a gel formulation of [14C]dapirivine for 7 days. Antimicrob Agents Chemother. 2008; 53:487–495. [PubMed: 19029331] 55. Nel AM, Coplan P, van de Wijgert JH, et al. Safety, tolerability, and systemic absorption of dapivirine vaginal microbicide gel in healthy, HIV-negative women. AIDS. 2009; 23:1531–1538. [PubMed: 19550287] 56. Nel AM, Coplan P, Smythe SC, et al. Pharmacokinetic assessment of dapivirine vaginal microbicide gel in healthy, HIV-negative women. AIDS Res Hum Retroviruses. 2010; 26:1181– 1190. [PubMed: 20854207] 57. Nel AM, Smythe SC, Habibi S, et al. Pharmacokinetics of 2 dapivirine vaginal microbicide gels and their safety Vs. hydroxyethyl cellulose-based universal placebo gel. J Acquir Immune Defic Syndr. 2010; 55:161–169. [PubMed: 20574411] 58. International Partnership for Microbicides. IPM Product Pipeline. Silver Spring, MD: http:// www.ipmglobal.org/products-development [accessed 15.01.12] 59. Soares EA, Santos AF, Sousa TM, et al. Differential drug resistance acquisition in HIV-1 of subtypes B and C. PLoS One. 2007; 2:e730. [PubMed: 17710130] 60. Schader SM, Colby-Germinario SP, Schachter J, et al. Synergy against drug-resistant HIV-1 with the microbicide antiretrovirals, dapivirine and tenofovir, in combination. AIDS. 2011; 25:1585– 1594. [PubMed: 21633286] 61. Romano J, Variano B, Coplan P, et al. Safety and availability of dapivirine (TMC120) delivered from an intravaginal ring. AIDS Res Hum Retroviruses. 2009; 25:483–488. [PubMed: 19388819] 62. Nel, A.; Habibi, S.; Smythe, S., et al. Pharmacokinetics and Safety Assessment of Monthly Anti- HIV DPV Vaginal Microbicide Rings with Multiple Dosing. 18th Conference on Retroviruses and Opportunistic Infections; February 27–March 2, 2011; Paper #1001 63. Microbicide Trials Network. [accessed 15.01.12] MTN-013/IPM 026: Phase 1 Safety and Pharmacokinetics of Dapivirine/Maraviroc Intravaginal Ring. http://www.mtnstopshiv.org/studies/ 2241 64. Selzentry (package insert). Research Triangle Park, NC: ViiV Healthcare; 2010. 65. ClinicalTrials.gov. A safety and pharmacokinetic study for dapvirine and maraviroc gel in Belgium. ClinicalTrials.gov; Identifier: NCT01242579; http://clinicaltrials.gov/ct2/show/ NCT01242579 [accessed 15.01.12] *66. Friend DR, Doncel GF. Combining prevention of HIV-1, other sexually transmitted infections and unintended pregnancies: development of dual-protection technologies. Antiviral Res. 2010; 885:s47–s54. [PubMed: 21109068] 67. Cole AM, Hong T, Boo LM, et al. Retrocyclin: a primate peptide that protects cells from infection by T- and M-tropic strains of HIV-1. Proc Natl Acad Sci U S A. 2002; 99:1813–1818. [PubMed: 11854483]

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68. Cole AM, Patton DL, Rohan LC, et al. The formulated microbicide RC-101 was safe and antivirally active following intravaginal application in pigtailed macaques. PLoS One. 2010; 5:e15111. [PubMed: 21124745] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 69. Sassi AB, Cost MR, Cole AL, et al. Formulation development of retrocyclin 1 analog RC-101 as an anti-HIV vaginal microbicide product. Antimicrob Agents Chemother. 2011; 55:2282–2289. [PubMed: 21321138] *70. Kashuba ADM, Patterson KB, Dumond JB, et al. Pre-exposure prophylaxis for HIV prevention: how to predict success. Lancet. 2011 epub ahead of print.

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Practice points

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript · An optimal microbicide formulation must be discrete, easy to use, safe for mucosal tissues, stable under a variety of climate conditions, and distribute evenly. · Product acceptability is critical for optimal adherence because no HIV prevention method will be effective if the target population does not find it easy to use. · Drug concentrations protective of HIV infection in mucosal tissues have yet to be determined; the pharmacokinetics and drug exposure will dictate optimal dosing regimens.

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Research agenda

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript · Pharmacokinetic exploration of differing results between CAPRISA 004 and VOICE. · Clinical studies of dapivirine gel and ring. · Drugs with mechanisms other than reverse transcription inhibition, such as entry inhibitors and integrase inhibitors. · Combination antiretroviral gel and ring products. · Continued efficacy end points with gel and ring formulations. · Development of unique formulations, such as films and tablets. · Combining HIV protection with contraceptive products.

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Table 1 Advantages and disadvantages of microbicide formulations. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Formulation Advantages Disadvantages Gels Female controlled. Effective compound must be stable in aqueous solution Discrete. Stability. Lubricating properties. Applicators required. Limited systemic absorption. Leaking is possible. Low side-effect potential. Frequent application may be required. Adherence disadvantage. Uneven distribution possible, depending on gel properties. Rings Female controlled. Sustained delivery must occur. Discrete. Distribution may not be uniform throughout genital No applicator required. compartment. No leakage May be uncomfortable or difficult to insert for some. Long term. More complex manufacturing. Adherence benefit. Limited systemic absorption. Low side-effect potential. Can be combined with contraception. Films and tablets Female controlled. Absorption may be dependent upon local hydration. Discrete. Local irritation could result from contact with solids. No applicator needed. Concern for uniformity of distribution. Viable option for drugs that would be degraded in aqueous solution. Could be formulated for immediate for timed release. No leakage. Low side-effect potential. Can be combined with contraception.

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Table 2 Human microbicide data for products in development. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Study Product/formulation Methods Efficacy and pharmacokinetic Tenofovir with or without emtricitabine Schwartz et al.40 Tenofovir 1% gel. Single and multiple dosing of Concentrations ranged from 1.2 × 104 to 9.9 × 106 ng/ once or twice daily 4 ml ml after a single dose, and 2.1 × 102 to 1.4 × 106 ng/ml applications for 2 weeks in 49 after multiple dosing in fluid samples with no difference women. in concentrations between proximal and distal tissues. Tenofovir diphosphate levels ranged from 7.1 × 103 to 8.8 × 106 ng/ml in endocervical cells, and drug concentrations were minimal in blood plasma with a median maximum concentration of 4.0 ng/ml after a single dose and 3.4 ng/ml after multiple dosing. Hendrix et al.42 Tenofovir 1% gel, Crossover design to compare African women preferred the gel form; American MTN 001 tenofovir oral tablet, or acceptability, tolerability and women preferred the oral form; most adverse effects both. pharmacokinetics in 144 were associated with oral tenofovir. Tissue women. Daily doses of either concentrations of the active tenofovir triphosphate were oral tenofovir, tenofovir gel, or 2352 fmol/mg after gel administration compared with both, were administered daily 17 fmol/mg after oral administration. Peripheral blood for 6 weeks in each period. mononuclear cell tenofovir–diphosphate concentrations were 52 fmol/million cells after oral administration and 5 fmol/million cells after gel administration. Anton et al.43 MTN Tenofovir 1% gel. 18 healthy participants received Rectal tolerability and acceptability was low after both 006 a single dose of oral tenofovir, single and multiple doses. Ex-vivo human then a single dose of either immunodeficiency virus challenge of rectal biopsies vaginally formulated gel used showed protection after multiple dosing with rectally rectally or placebo gel rectally applied tenofovir gel compared with placebo, but not (2:1), followed by 7 days of single gel dosing. daily gel dosing. Abdool Karim et Tenofovir 1% gel. 843 women used one Tenofovir was 39% effective at preventing HIV al.26 CAPRISA004 application (4 ml) of gel up to infection with average adherence and 54% effective in 12 h before and after sex for an the highest of adherers. The tenofovir concentrations average of 18 months of follow correlated with likelihood of infection, with women up. with cervicovaginal fluid concentrations greater than 1000 ng/ml more likely to be protected and less than 1000 ng/ml less likely to be protected. VOICE49 Tenofovir 1% gel; Once-daily dosing in 5000 Gel arm stopped early due to futility in November 2011 tenofovir 300 mg oral women (approximately 1000 after 2 years of follow up. Full results expected in early tablet; tenofovir plus women in the gel arm). 2013. emtricitabine oral tablet. FACTS50 Tenofovir 1% gel. Approximately 2200 women Trial is ongoing. randomly assigned to tenofovir gel or placebo gel used one application of gel up to 12 h before and after sex. Dapivirine Nel et al.55 Dapivirine 0.001%, 2.5 ml applied vaginally twice All doses were found to be safe, with dose proportional 0.002%, 0.005%, 0.02% daily for 42 days in 119 women. plasma concentrations across the dosing range. The gel. maximum observed plasma concentration was 474 pg/ ml in the 0.02% dose group. Nel et al.56 Dapivirine 0.001%, 2.5 ml applied vaginally in 18 All doses were safe and well tolerated. Mean peak 0.005%, 0.02% gel. women for 10 days; once daily values in cervicovaginal fluid were 4.6–8.3 × 106 pg/ml on days 1 and 10 and twice on day 1 and 2.3–20.7 × 106 pg/ml on day 10. daily on days 2–9. Dapivirine concentrations in vaginal tissue biopsies collected at day 10 were 1.0–356 × 103 pg/mg. Nel et al.57 Dapivirine 0.05% gel. 2.5 g applied vaginally daily for Cervicovaginal fluid concentrations were at least 5 logs 11 days in 36 women. higher than the in-vitro inhibitory concentrations and maximum plasma concentrations did not exceed 1.1 ng/ ml. Romano et al.61 Dapivirine 25 mg, and 25 women inserted the ring for Both concentrations of rings were found to have 200 mg ring. 7 days, with blood plasma and concentrations throughout the genital tract that vaginal fluid sampling from the exceeded 1000 times the EC50. The lowest introitus, cervix, and ring area. concentration in any of the tissue samples was over 30-

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Study Product/formulation Methods Efficacy and pharmacokinetic fold higher than the in-vitro EC90. Mean plasma

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript concentrations were less than 50 pg/ml. Nel et al.62 Dapivirine 25 mg Ring. 48 women randomised (3:1) to Dapivirine drug concentrations in vaginal fluid peaked dapivirine ring or placebo ring. within 24 h remained consistent for up to 35 days at Group A inserted a first ring for levels well above the IC50, with even distribution 28 days then a second ring on throughout the genital tract. The maximum plasma day 31 for another 28 days. concentration measured was 553 pg/ml. Group B inserted a first ring for 35 days, then a second ring on day 38 for 21 days, then a third ring on day 59 for 24 h.

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